Use of 4-cholesten-3-one as an anti-obesity agent

ABSTRACT

The present invention provides a method for using the anti-obesity agent comprising 4-cholesten-3-one represented by the following formula: ##STR1## as an effective component. The anti-obesity agent of the present invention is useful because of inhibition effect of body weight increase and body fat accumulation. The present invention also provides a medicinal composition comprising 4-cholesten-3-one as an effective component and a pharmaceutically acceptable excipient as well as methods for preventing and treating obesity comprising administering an effective amount of 4-cholesten-3-one to a person.

TECHNICAL FIELD

The present invention relates to an anti-obesity agent and morespecifically to an anti-obesity agent comprising cholest-4-en-3-one(4-cholesten-3-one) as an effective component.

BACKGROUND ART

The obesity means such a body condition that adipose tissue (body fat)is systemically overaccumulated and results from an energy intake higherthan the energy expenditure over a long period of time. The obesity isaccompanied by the limitation in the physicalactivity and theapplication of an excess load to various internal organs and results invarious associated diseases such as arteriosclerosis, cancers anddiabetes mellitus to thus ruin the obese person's health.

Until now, there have been proposed a variety of medicinal-therapeuticmethods for preventing obesity, but these methods must solve variousdifficulties in order to ensure a satisfactory effect. For instance, thedrug therapy using hormones or excitometabolics suffers from a problemthat the decomposition of body proteins are promoted simultaneously withthe decomposition of body fats. Among the hormone, a certain androgenichormone has been known to show anti-obesity effects through thestimulation of the myotropic action and hence the promotion of theconsumption of reserve fats. Examples of such androgenic hormonesinclude dehydroepiandrosterone and 3-keto-Δ↑9-19-norsteroid (JapaneseUn-examined Patent Publication No. Hei 02-275895). In addition,anorectics and digestive enzyme-inhibitors suffer from a problem ofside-effects such as nervous sympton and diarrhea.

Accordingly, an object of the present invention is to provide ananti-obesity agent free of the foregoing disadvantages.

Another object of the present invention is to provide a medicinalcomposition effective for the prevention and treatment of obesity.

A still another object of the present invention is to provide a methodfor preventing obesity and treating patients suffering from obesity.

DISCLOSURE OF THE INVENTION

The inventor of this invention has found out that 4-cholesten-3-one hasa fat accummulation-inhibitory effect and that obesity can be preventedand treated through the administration of this compound, and thus havecompleted the present invention.

According to an aspect of the present invention, there is provided ananti-obesity agent which comprises 4-cholesten-3-one as an effectivecomponent and which is effective for the prevention and treatment ofobesity.

According to another aspect of the present invention, there is provideda medicinal composition which comprises 4-cholesten-3-one as aneffective component and a pharmaceutically acceptable excipient andwhich is effective for the prevention and treatment of obesity.

According to a still another aspect of the present invention, there isprovided a method for preventing obesity and for treating a patientsuffering from obesity, characterized in that an effective amount of4-cholesten-3-one is administered to a person.

The anti-obesity agent of the present invention is quite useful since itis substantially non-toxic.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows time-course in the averaged body weight of CDF1 mice ineach group.

FIG. 2 shows the relation between the monthly age and the survival rateof CDF1 mice in each group.

FIG. 3 shows the averaged weights of the organs, i.e., brain, lung,heart, liver, kidneys, spleen and testes (or ovaries) of CDF1 mice of18-month-old in each group.

FIG. 4 shows the averaged weights of the organs, i.e., pituitary glandand adrenal gland of CDF1 mice of 18-month-old in each group.

FIG. 5 shows the averaged amount of fats present in abdominal adiposetissues of CDF1 mice of 18-month-old in each group.

FIG. 6 shows time-course in the averaged body weight of CDF1 mice ineach group.

FIG. 7 shows the averaged values of serum lipids of CDF1 mice in eachgroup.

BEST MODE FOR CARRYING OUT THE INVENTION

4-Cholesten-3-one may be chemically synthesized or may be producedstarting from cholesterol as a substrate using cholesterol oxidase(cholesterol:oxigen oxidoreductase: EC1, 1, 3, 6) produced byBrevibacterium sp., Cullulomonas sp., Nocardia erythropolis, Pseudomonasfluorescens, Schizophyllum commune, Streptomyces sp., Mycobacteriumcholesterolicum or other microorganisms. Moreover, it is also possibleto use commercially available ones produced by either of the foregoingmethods.

The compound, 4-cholesten-3-one, is represented by the following formula(I) and has the following physical and chemical properties: ##STR2## (1)Molecular Formula: C₂₇ H₄₄ O (2) Molecular Weight: 384.65

(3) Melting Point (mp): 82° C.

(4) Specific Rotatory Power, [α]_(D) :+88° (in chloroform)

(5) UVλ_(max) (mn): 240,320

(6) Solubility: The compound is hardly soluble in water; slightlysoluble in alcohol; soluble in ether, chloroform, pyridine, benzene andpetroleum ether; and also highly soluble in fats and oils.

(7) Taste.Odor.Color: The compound is a stable tasteless, odorless andachromatic crystal at ordinary temperature.

(8) CAS Number: CAS [601-57-0]

While not wanting to be bound by any particular theory, the mechanismwhereby the compound 4-cholesten-3-one shows the anti-obesity effectwill be assumed to be as follows. The fats ingested in the form of dietsare taken in the interior of the spherical membrane of cylomicron, i.e.,a lipoprotein in the small intestine and are then transferred to adiposetissues. On the other hand, the fats synthesized in the liver are takenin the interior of the spherical membrane of a VLDL (very low densitylipoprotein) and then transferred to adipose tissues. These lipoproteinshaving a fat-transporting function such as cylomicron and VLDL each isformed from a membrane which comprises three components, i.e.,cholesterol, phospholipids and apoproteins and serves to take lipids,i.e., triglyceride and cholesterol esters therein and to transport themto each tissue. As has been explained above, cholesterol is a principalconstituent of the lipoprotein having a fat-transporting function.4-Cholesten-3-one antagonizes the action of the cholesterol, thusinhibits the formation of the foregoing lipoprotein and, in turn, thetransportation of the lipids and accordingly, shows the inhibitioneffect of body fat accumulation. As a result, the compound ensures theobesity-inhibitory effect. Alternatively, it is also assumed that4cholesten-3-one inhibits the formation of adrenal corticoid through theantagonistic action to that of the cholesterol and, in turn, inhibitsthe formation of fatty acids in the liver and as a result, the compoundshows its anti-obesity effect.

Food products can be prepared using the anti-obesity agent of thepresent invention. When incorporating the agent into food products,4-cholesten-3-one may directly be added to each food and alternatively,the compound is first added to fats and oils as starting materials foran intended food and then the food is prepared from the fats and oils.4-Cholesten-3-one may be added to a food or fats and oils in an amountranging from 1 to 5,000 mg per 100 g of foods or fats and oils.Moreover, 4-cholesten-3-one can be formulized into variouspharmaceutical formulations such as solutions, suspensions, capsules,powder, granules, grains or tablets (pills) and these formulations maythen be added to foods or fats and oils.

The anti-obesity agent of the present invention may also be formulatedinto medicinal preparations. In this case, the formulations may beadministered through any route. They may be administered through, forinstance, an oral, intravenous, intraperitoneal, subcutaneous orintramuscular route, with the administration through the oral routebeing preferred. In case of oral administration, 4-cholesten-3-one maybe administered alone or in combination with pharmaceutically acceptableexcipients in the form of various preparations such as solutions,suspensions, powder, granules, capsules or tablets. The excipients maybe those commonly used, for instance, sugars such as lactose, sucroseand glucose; starches; inorganic substances such as calcium carbonateand calcium sulfate; crystalline cellulose, distilled water, purifiedwater, sesame oil, soybean oil, corn oil, olive oil and cotton seed oil.When formulating the compound into preparations, various additives canbe used and examples thereof include binders, lubricants, dispersants,suspending agents, emulsifiers, diluents, buffers, antioxidants andgermicides. Moreover, appropriate buffers, isotonic solutions or thelike may be added to the compound and then the resulting mixture isdissolved in, for instance, oils such as vegetable oils to giveinjectable solutions. 4-Cholesten-3-one may also be mixed with othermedicines or may be used in combination therewith. In this respect, theforegoing pharmaceutical preparations may be subjected to asterilization treatment.

The dose of the anti-obesity agent of the present invention variesdepending on, for instance, ages, sexes, clinical sign, administrationpathway, administration times per day, dosage forms, but the dose foradult desirably ranges from about 1 to 1,500 mg/kg body weight/day interms of the weight of 4-cholesten-3-one, for the oral administration.

The present invention will hereunder be explained in more detail withreference to the following examples. These examples never limit thescope of the present invention, but are given for illustrating thepresent invention.

(Example 1)

A. Test Method

1. Experimental Animals and Care Conditions

Male and female CDF1 mice (BALB/C×DBA/2 F1) of 4-week-old (availablefrom Charles River Japan Inc.; 300 animals in all (150 males and femaleseach).) were divided into three groups, i.e., 4-cholesten-3-one fedgroup, cholesterol fed group and untreated control group (each groupbeing divided into two subgroups each comprising 50 male or 50 femaleanimals). The following test was carried out using these 6 subgroups.

All of the mice were accommodated in aluminum cages (5 animals per cage)and bred for 17 months in an animal room at a temperature of 24°±1° C.and a relative humidity of 55±5% and under 12:12 hr light/dark schedule.The cages and the bedding (White flake) were replaced with fresh onesevery two days.

2. Preparation of Experimental Feed

The following test feeds 1) to 3) were prepared using a high fatcommercial feed for breeding (type CMF, Oriental Yeast Co., Ltd., Tokyo)as a basal diet, formed into pellets and supplied to hoppers. Tap wateras drink water was introduced into water bottle and the animals wereallowed feed and water ad libitum. When the following feed 1) and 2) wasused, the doses of 4-cholesten-3-one and cholesterol per mouse wereabout 650 mg/kg body weight/day, respectively.

1) Feed for the 4-Cholesten-3-One Fed Group: 4-Cholesten-3-one(available from Aldrich Chemical Co., USA) was added to the basal dietin an amount of 0.5% by weight based on the weight of the basal diet.

2) Feed for the Cholesterol Fed Group: Cholesterol (available fromAldrich Chemical Co., USA) was added to the basal feed in an amount of0.5% by weight based on the weight of the basal feed.

3) Feed for the Untreated Control Group: Any additive was not added tothe basal feed.

The CMF feed has the following composition: crude proteins 29.4%; crudefats 8.7%; minerals 6.5%; crude fibers 3.4%; soluble nitrogen-freeextract 43.6%; and it has a moisture content of 8.4% and a calory of 370Kcal/100 g. This is a feed having a fat content and a calory higher thanthose of the commercial feed which has a crude fats content of 4.4% anda calory of 343 Kcal/100 g.

3. Items to be Inspected

(1) Determination of Changes in Body Weights

All of the surviving mice were examined for the body weights everymonths followed by calculation of the average value thereof for eachgroup.

(2) Determination of Survival Rate

The number of surviving animals were determined every months tocalculate the surviving rate. Moreover, the mice died were subjected tonecropsy to inspect for tumor incidence and other lesions and theresults obtained were recorded.

(3) Determination of Organ Weight

After 17 months of feeding, all the mice were sacrified throughanesthetizing them with carbon dioxide and animals free from any lesionwere anatomized to inspect for tumor incidence and other lesions. Inanimals free from lesions, the weights of brain, lung, heart, liver,kidneys, spleen, testes (or ovaries), pituitary gland and adrenal gland,were measured and the average value thereof for each group wascalculated.

(4) Determination of Amount of Abdominal Adipose Tissue

After 17 months of feeding, animals free from any lesion were examinedfor the amount of abdominal adipose tissue, followed by calculation ofthe average values thereof for each group.

(5) Determination of Rate of Tumor Incidence

After 17 months of feeding, all the mice were sacrified throughanesthetizing them with carbon dioxide and the number of animalssuffering from tumor was scored.

B. Test Results

(1) Determination of Changes in Body Weights

Time-course of the averaged body weight of each group is plotted on FIG.1 together with the reference data. Each value plotted on FIG. 1 isexpressed in terms of the average value±standard deviation. The symbol:means that the probability (p) in the result of the student t-test isless than 0.05 and the symbol: means that p is less than 0.001. Onemonth after the initiation of the experiment, the body weights of the4-cholesten-3-one fed groups (both male and female groups) were found tobe lower than those observed for the untreated control groups and thedifference therebetween was increased as the time proceeded. After 4months, the body weight of the 4-cholesten-3-one fed group (malesubgroup) was 13% smaller than that of the control group and thereafterthe difference therebetween varied between 13 and 15%, while the bodyweight of the 4-cholesten-3-one fed group (female subgroup) was 12%smaller than that of the control group after 6 months and the differencetherebetween then varied between 12 and 14%.

The body weights of the cholesterol fed groups (both male and femalegroups) were almost identical to those observed for the untreatedcontrol groups, but they were apt to increase as the time proceeded, ascompared with those of the control groups.

The reference data for male animals are those reported by Morisada etal. (Jpn. J. Cancer Res., 1989, 80, pp. 77-82), i.e., the averaged bodyweights of the CDF1 mice (available from Charles River Japan Inc.) whichwere bred and raised up to 4-month-old using the commercial stock feed.These data are almost identical to those observed for the4-cholesten-3-one fed groups.

The male and female mice in the 4-cholesten-3-one fed groups werenormally raised in their appearance, while the mice of the cholesterolfed and untreated control groups had clearly corpulent figures.

The foregoing results indicate that the body weights of the cholesterolfed and untreated control groups were increased obesely due to feedingof high energy foods, while the 4-cholesten-3-one fed groups werenormally raised and bred.

(2) Determination of Survival Rate

The mortality curve observed for each group is shown in FIG. 2.

There is not observed any significant difference in the survival ratebetween the groups tested (including both male and female subgroups) upto 18-month-old.

This fact indicates that the feeding of 4-cholesten-3-one does notaffect the mortality and that it is believed to be non-toxic.

(3) Determination of Organ Weight

The averaged weights of these organs observed for each group are plottedon FIGS. 3 and 4.

There was not any significant difference in the weight of each organ,i.e., brain, lung, heart, liver, kidneys, spleen or testes (or ovaries)between the groups (including both male and female subgroups). Moreover,there was not likewise observed any significant difference in theweights of the hormone-producing organs, i.e., pituitary gland andadrenal gland, between the groups (including both male and femalesubgroups).

As seen from these facts, the feeding of 4-cholesten-3-one did notadversely affect the growth and functions of each organ.

(4) Determination of Amount of Abdominal Adipose Tissue

The averaged amount of the abdominal adipose tissue observed for eachgroup is shown in FIG. 5. Each value in FIG. 5 is expressed in terms ofthe average value±standard deviation. The symbol: means that theprobability (p) in the result of the student t-test is less than 0.05and the symbol: means that p is less than 0.01.

The averaged amount of the abdominal adipose tissue observed for the4-cholesten-3-one fed group was about 1/3 time those observed for theuntreated control and cholesterol fed groups for the male subgroups andabout 1/2 time those observed for the untreated control and cholesterolfed groups for the female subgroups.

In addition, the untreated control and cholesterol fed groups hadsubcutaneous fats of back and abdominal regions, while the4-cholesten-3-one fed group had only a slight subcutaneous fats of theseregions (data are not shown). It can be concluded that the feeding of4-cholesten-3-one inhibits the body fat accumulation in abdominal andsubcutaneous and prevents obesity.

(5) Determination of Rate of Tumor Incidence

The numbers of tumorous incidence formed in animals of each group arelisted in the following Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Tumor Incidence in CDF1 Mice                                                               Number of mice with tumor                                                     No. of                                                                             Small           Sexual  Total                               Group  Sex   animals.sup.a)                                                                     Intestine                                                                          Lung                                                                             Liver                                                                             Spleen                                                                            Organ                                                                             others                                                                            Number                                                                             Lymphoma                       __________________________________________________________________________    4-cholesten-3-                                                                       male  34    .sup. 17(50).sup.b)                                                               6  2   0   0   1   19(56)                                                                             1                              one fed group                                                                        female                                                                              45   20(44)                                                                             0  0   2   1   0   20(44)                                                                             3                              cholesterol                                                                          male  34   15(44)                                                                             8  3   2   0   0   18(52)                                                                             0                              fed group                                                                            female                                                                              46   14(30)                                                                             1  1   2   3   0   17(37)                                                                             2                              un-treated                                                                           male  35   24(69)                                                                             9  0   1   0   0   26(74)                                                                             1                              control group                                                                        female                                                                              47   17(36)                                                                             5  1   0   5   1   23(49)                                                                             2                              __________________________________________________________________________     .sup.a) Number of mice suviving month on 18;                                  .sup.b) The numerical value in the parentheses represents percentage.    

In either of the groups, the small intestinal polyposis (one of benigntumors) are observed in a high incidence which is not less than 40% formale mice and not less than 30% for female mice, but any significantdifference in the incidence between the groups tested was not observedat all. In respect of tumors formed in sites such as lung, liver,spleen, sexual organs and other sites, there was not observed anycharacteristic tendency in every groups. Moreover, the overall incidenceof tumors for the male mice was greater than that observed for thefemale mice, but any significant difference in the incidence between thegroups tested was not observed at all.

It can be concluded, from the foregoing facts, that the feeding of4-cholesten-3-one (about 650 mg/kg body weight/day) does not show anycarcinogenicity and tumor promoting activity. It has been reported thatthe small intestinal polyposis are developed at rates of 51% and 37% formale and female BALB/c mice of 12-month-old, the female BALB/c micebeing the mothers of the CDF1 mice (Mizutani et al., Cancer Lett., 1984,25, pp. 19-23). Accordingly, the incidence of small intestinal polyposiswould be high in all of the groups tested for the reason that thepolyposis would be inherited in the CDF1 mice.

Furthermore, there was not observed any characteristic difference,between the groups tested, in the development of diseases or lesionsother than tumors.

(Example 2)

A. Test Method

1. Experimental Animals and Care Conditions

Male and female CDF1 mice of 5-week-old (available from Charles RiverJapan Inc.; 60 animals in all) were divided into six groups (30 animalseach), i.e., male and female groups to which the common feed was fed(hereunder referred to as "common feed fed group"), male and femalegroups to which a feed having a high fat content was fed (hereunderreferred to as "high fat feed fed group") and male and female groups towhich a feed having a high fat content and containing 4-cholesten-3-onewas fed (hereunder referred to as "4-cholesten-3-one fed group"), eachgroup comprising 10 mice. All of the mice were accommodated in aluminumcages (5 animals per cage) and bred for 13 days in an animal room at atemperature of 24°±1° % C. and a relative humidity of 50±5% and under12:12 hr light/dark schedule. The cages and the bedding were replacedwith fresh ones every two days.

2. Preparation of Experimental Feed

The following test feeds 1) to 3) (mash feeds) were prepared using amodified AIN-formula-purified diet (Oriental Yeast Co., Ltd., Tokyo) asa basal diet and supplied to hoppers. Tap water as drink water wasintroduced into water bottle and the animals were allowed feed and waterad libitum.

1) Feed for Stock Feed Fed Group: The basal feed as such was used. Thefat content thereof was found to be 6%.

2) Feed for High Fat Feed Fed Group: Soybean oil was added to the basalfeed in an amount of 11.5 g per 100 g of the latter to adjust the fatcontent thereof to 15%.

3) Feed for 4-Cholesten-3-One Fed Group: To the feed for the high fatfeed fed group, there was added 4-cholesten-3-one (available fromAldrich Chemical Co., USA) to a concentration of 1%. The fat contentthereof corresponds to 14.8%.

The modified AIN-formula-purified diet has the following composition:corn starch 41.5%; casein 25%; α-starch 10%; cellulose powder 8%;soybean oil 6%; minerals (AIN-76) 3.5%; granulated sugar 5%; andvitamins (AIN-76 and choline bitartrate) 1%.

3. Items to be Inspected

(1) Determination of Changes in Body Weights

The mice were examined for the body weights at 6th and 13th day,followed by calculation of the average value thereof for each group.Each feed was replaced with fresh one every two days and at this stage,the remaining feed was weighed to calculate the amount of feed intake.

(2) Autopsy and Analysis of Lipids in Serum

After breeding over 13 days, all of the mice were sacrified using carbondioxide and then the autopsy and the analysis of lipids in the sera werecarried out.

B. Test Results

(1) Determination of Changes in Body Weights

The variation of the averaged body weight of each group is plotted onFIG. 6. Each value plotted on FIG. 6 is expressed in terms of-theaverage of 10 animals in each group±standard deviation. The symbol:means that the probability (p) in the result of the student t-test isless than 0.05 and the symbol: means that p is less than 0.001. Thecommon feed fed group and the high fat feed fed group showed constantincreases in the body weights, while the 4-cholesten-3-one fed group didnot show any increase in the body weight and the body weights thereofwas rather apt to decrease. In case of the male mice, there was soonobserved a statistically significant difference (p<0.05), even after 6days, between the 4-cholesten-3-one fed group and the other two groups.In case of the female mice, the same phenomenon as that for the malemice was likewise observed, but the difference increased gradually overthe entire test period.

The rates of feed intake, as expressed in terms of the weight(g)/animal/day, were found to be 4.20 for the stock feed fed group, 4.25for the high fat feed fed group and 4.38 for the 4-cholesten-3-one fedgroup, respectively and were thus approximately identical to oneanother. The amount of 4-cholesten-3-one fed to the corresponding groupwas calculated on the basis of the rate of feed intake thus determinedand found to be about 2,100 mg/kg body weight/day.

The mice in either of the test groups were visually active and therewere not any clinical signs such as coat roughness and diarrhea.

(2) Autopsy and Analysis of Lipids in Serum

As a result of the autopsy, there were not observed any abnormalitiessuch as hypertrophy, atrophy and color tone change of the major organs,i.e., lung, heart, liver, kidneys, spleen and testes (or ovaries) aswell as adrenal glands in all of the groups tested.

The results of the analysis of serum lipids are shown in FIG. 7. Eachvalue shown in FIG. 7 is expressed in terms of the average of 10 animalsin each group±standard deviation. The symbol: means that the probability(p) in the result of the student t-test is less than 0.05, the symbol:means that p is less than 0.01 and the symbol: means that p is less than0.001. The triglyceride content in the serum was decreased in the4-cholesten-3-one fed group and this tendency was particularlyconspicuous in the male mice. On the other hand, the total cholesterolcontent in the serum observed for the 4-cholesten-3-one fed group wasslightly greater than that observed for the stock feed fed group and wasalmost identical to that observed for the high fat feed fed group. Thephospholipid values in the sera observed for the 4-cholesten-3-one fedgroup and the stock feed fed group were approximately identical to oneanother for the male mice, while those observed for the female mice wereslightly higher than those observed for the male mice, but there was notobserved any statistically significant tendency.

The foregoing indicates that the body weights of the CDF1 mice were notincreased, but decreased through addition of 1% 4-cholesten-3-one to thefeed and that the triglyceride values in the sera of the CDF1 mice werealso reduced. It was also found that the body weight-controlling effectof 4-cholesten-3-one was independent of the difference in the amount ofthe feed intake. Moreover, 4-cholesten-3-one was found to besubstantially non-toxic.

INDUSTRIAL APPLICABILITY

The anti-obesity agent of the present invention is useful because ofinhibition effect of the body weight increase and body fat accumulation.Moreover, the anti-obesity agent of the present invention issubstantially non-toxic and can inhibit the body fat accumulation and inturn can prevent or treat obesity even when high energy foods are taken.The anti-obesity agent of the invention is also useful since theprevention or treatment of the obesity with the anti-obesity agent ofthe present invention would permit the prevention of various diseasessuch as arteriosclerosis, cancers and diabetes mellitus associated withthe obesity.

I claim:
 1. A method for preventing obesity comprising administering aneffective amount of 4-cholesten-3-one to a person in need thereof.
 2. Amethod for treating obesity comprising administering an effective amountof 4-cholesten-3-one to a person.